1. Field of the Invention
The present invention relates to phosphite-borane derivatives with nucleoside substituents that exhibit antineoplastic, anti-hyperlipidemic, and anti-inflammatory activity.
2. Description of the Related Art
A. Lewis Base-borane Compounds
Various boron-containing compounds have previously been shown to exhibit therapeutic biological activity. For example, amine-borane compounds such as amine.BH.sub.2 COOH, amine.BH.sub.2 COOMe and amine.BH.sub.2 CONHR have been demonstrated to exhibit antitumor, anti-inflammatory and hypolipidemic activities. Additionally, phosphite-borane compounds have been used in hydroboration under mild conditions (Pelter, A., et al, J. Chem. Soc. Chem. Commun. 1981, 1014). Since the first reports of phosphite-borane compounds and their properties (Reetz, T., J. Am. Chem. Soc. 1960, 82, 5039), very few phosphite-borane compounds have been synthesized and/or had their properties investigated (Das, M. K., et al, Synth. React. Inorg. Met. Org. Chem. 1986, 16, 67; Martin, D. R. et al; Pennington, B. T., J. Inorg. Nucl. Chem. 1978, 40, 9; and Mutterties, E. L., "The Chemistry of Boron and its Compounds," Wiley, New York, 1967).
Generally, phosphite-borane derivatives may be considered as analogs of alkylphosphates, (RO).sub.3 P.dbd.O vs. (RO).sub.3 PBH.sub.3, as well as analogs of alkylphosphonates, e.g., (RO).sub.2 P(O)CH.sub.3 vs. (RO).sub.2 P(O)BH.sub.3, or (RO).sub.2 P(O)CH.sub.2 X vs. (RO).sub.2 P(O)BH.sub.2 X, wherein R is alkyl and X is heteroatom substituent. Since phosphate and phosphonate groups are present in a variety of biologically important molecules, e.g., DNA, RNA, phospholipids, aminophosphonates, etc., their boron-containing analogs may prove useful as biomolecular probes and as potential therapeutic agents.
Additionally, several synthetic phosphonates, e.g., phosphonoacetic acid, phosphonoformic acid, etc., have been found to possess significant antiviral activity (Mayer, R. F., et al, Antimicrob. Agents Chemother. 1976, 9, 308; Oberg, B., Pharmac. Ther., 1983, 19, 387; and Clerq, E. D., J. Med. Chem. 1986, 29, 1561). This antiviral activity coupled with the established pharmacological activity of amine-borane derivatives makes phosphite-borane derivatives potentially significant as a class of bioactive compounds.
B. Modified Nucleotides
Ribo- and deoxyribonucleoside 5'-mono-, di-, and triphosphates play a central role in the metabolism of nucleic acids, one of the most important polymer molecules of living systems. It has long been realized that chemically modified analogs of nucleoside mono-, di-, and tri-phosphates may be useful tools to probe different steps of nucleic add metabolism. It has also been recognized that they may have valuable chemo-therapeutic properties. Therefore, synthesis and study of nucleotide analogs has long been in the center of interest.
Several modifications of the phosphate group have been carried out and the derivatives are shown in Table 1 below. These derivatives mainly involve phosphorothioates (Eckstein, F. Angew Chem. Int. Ed. Engl. 1983, 22, 423-439 and references therein, Eckstein, F. Ann. Rev. Biochem. 1985, 54, 367-402 and references therein, Ludwig, J.; Eckstein, F. J. Org. Chem. 1989, 54, 631-635, and Ludwig, J.; Eckstein, F. J. Org. Chem. 1991, 86, 5860-5865), phosphorodithioates (Ludwig, J.; Eckstein, F. J. Org. Chem. 1991, 56, 1777-1783), phosphoramidates (Chambers, R. W.; Moffatt, J. G., J. Am. Chem. Soc. 1958, 80, 3752-3756; Chambers, R. W. et al, ibid, 1960, 82, 970-975; Moffatt, J. G.; Khorana, H. G., ibid, 1961, 83, 649-658; Cramer, F. et al, Chem. Ber. 1961, 94, 1612-1621; Schaller et al. ibid, 1961, 94, 1621-1633; Cramer, F.; Neunhoffer, H., ibid, 1962, 95, 1664-1669; Simoncsits, A.; Tomasz, J., Tetrahedron Lett. 1976, 3995-3998; Tomasz, J.; Simoncsits, A., J. Carbohydrates--Nucleosides--Nucleotides 1978, 5, 503-522; Tomasz, J., Nucleosides & Nucleotides 1983, 2, 63-79; Bakina, G. T. et al. Bioorg. Khim, 1975, 1, 611-615 and Zarytora, V. F. et al, ibid 1975, 1, 793-798), phosphonates (Anand, N.; Todd, A. R., J. Chem. Soc. 1951, 1867-1872; Engel, R. Chem. Revs. 1977, 77, 349-367 and references therein ad Myers, T. C.; Simon, N. L., J. Org. Chem. 1985, 30, 443-446), phosphorofluoridates (Wittmann, R., Chem. Ber. 1963, 96, 771-779, Johnson, P. W. et al, Nucleic Acids Res. 1975, 2, 1745-1749; Staley, B.; Yount, R. G., Biochemistry 1972, 11, 2863-2871 and Eckstein, F. et al, ibid, 1975 114, 5225-5232), phosphites or H-phosphonates (Corby, N. S. et al, J. Chem. Soc., 1952, 3669-3674, Sir Todd, A., ibid, 1961, 2316-2320 and Holy, A. et al, Cell. Czech. Chem. Commun. 1965, 30, 1635-1641), phosphorazidates (Chladek, S. et al., Biochemistry 1977, 16, 4312-4319), phosphonoselenoates (Sekine, M.; Hata, T., Tetrahedron Lett. 1979, 801-802) and alkyl phosphates (Hoffmann, P. J.; Blakley, R. L., Biochemistry 1975, 14, 4804-4812.
TABLE 1 __________________________________________________________________________ Structural formulae of nucleoside 5'-mono-, di- and triphosphates derivatised at the phosphorus ##STR2## ##STR3## ##STR4## X Y X Z Y X __________________________________________________________________________ 1 S.sup.- 2 O.sup.- S.sup.- 4 O.sup.- O.sup.- S.sup.- 10 NH.sub.2, NHR' or NR'.sub.2 3 S.sup.- O.sup.- 5 O.sup.- S.sup.- O.sup.- 13 CH.sub.3 or CH.sub.2 R' 14 CH.sub.3 O.sup.- 6 S.sup.- O.sup.- O.sup.- 15 F 16 F O.sup.- 7 O.sup.- S.sup.-.sub.3 S.sup.- 18 H 20 N.sub.3 O.sup.- 8 S.sup.- O.sup.- S.sup.- 19 N.sub.3 11 O.sup.- O.sup.- NH.sub.2 22 Se.sup.- 12 R'NH O.sup.- O.sup.- ##STR5## 17 21 23 F N.sub.3 R'O O.sup.- O.sup.- O.sup.- O.sup.- O.sup.- O.sup.- __________________________________________________________________________
Derivatization at the phosphorus moiety by replacing a non-bridging oxygen atom confers chirality on the P1 phosphorus of compounds 2, 4, 7, 8, and 11 as well as P2 of 5 and 7. As a result of the chirality of the sugar residue, nucleotide 5'-di- and triphosphate derivatives 2, 4, 5, 7, 8 and 11 formed during chemical synthesis exist as pairs of phosphorus, Rp and Sp, diastereoisomers. The chirality of phosphorus renders these derivatives suitable tools for studying the stereochemistry of enzyme catalyzed reactions. The diastereoisomers, however, have to be separated, since the diastereoisomeric purity of the substrate is an essential prerequisite for stereochemical studies.
Taking into consideration the chirality of phosphorus, it is not surprising that, among the nucleoside 5'-mono-, di-, and triphosphate analogs listed in Table 1 above, the thiophosphates have found widespread applications in biochemistry and molecular biology. Nucleoside phosphorothioate diastereoisomers have been used to determine the stereochemical course of numerous enzyme catalyzed nucleotidyl and phosphoryl transfer reactions (Eckstein, F., Angew Chem. Int. Ed. Engl. 1983, 22, 423-439 and references therein and Eckstein, F. Ann. Rev. Biochem. 1985, 54, 367-402 and references therein). The stereochemical outcome of an enzymic reaction, i.e., whether it proceeds with inversion or retention of configuration at phosphorus, is an informative criterion about the presence or absence of a covalent enzyme intermediate. The Sp diastereoisomers of 4, as substrates of RNA and DNA polymerases, have successfully been employed for sequencing (Gish, G.; Eckstein, F., Science, 1988, 240, 1520-1522 and Nakamaye, K. L. et al., Nucleic Acids Res. 1988, 16, 9947-9959), oligonucleotide-directed mutagenesis (Nakamaye, K. L.; Eckstein, F., Nucleic Acids Res., 1986, 14, 9679-9698 and Sayers, J. R. et al, ibid, 1988, 16, 791-802), and the labeling of the hybridization probes (Haase, A. T. et al, Science 1985, 227, 189-192 and Bahmanyar, S. et al, Science, 1987, 237, 77-80). Triphosphate derivatives 6 are also substrates for polymerases (Smith, M. M. et al, Biochemistry 1978, 17, 493-500). Many enzymes show strong preference for either the Sp or Rp diastereoisomer of triphosphates 4 and 5. For example, of guanine nucleotide-binding proteins (G-proteins) which are implicated in signal transduction pathways (Bourne, H. R. et al, Nature 1990, 348, 125-132), transducin (the G-protein involved in vision) has a stronger affinity for the (Sp)-guanosine 5'-0-(2-thiotriphosphate). On the other hand, the G-protein responsible for the oscillatory release of Ca.sup.2+ ions in most cells is preferentially activated by the (Rp)-diastereoisomer (von zur Muhlen, R.; Eckstein, F.; Penner, R. Proc. Acad. Sci. U.S.A. 1991, 88, 926-930). The stereoselectivity of the enzymes can be reversed by changing the metal cation necessary for the enzyme action from a hard to a soft one (Armstrong, V.; Eckstein, F., Eur. J. Biochem., 1976, 70, 33-38).
Nucleoside-boranophosphates and boranophosphoramidates (phosphite-borane compounds), the compounds of the present invention, may be considered as analogs of corresponding phosphates or thiophosphates, where the oxygen or sulfur has been replaced with a borane substituent. These derivatives have similar charges and thus resemble phosphates or phosphorothioates.
On the other hand, differences are expected between boranophosphates and thiophosphates in reactivity, hydrogen bonding and metal ion chelating ability which may be a determinant for enzyme reactions. Consequently, it seems reasonable to suppose that boranophosphates may find similar and, at the same time, complementary applications to thiophosphates 1-6. Our initial results support this supposition.
The thymidine 5'-O-(1-boranotriphosphate) can substitute for thymidine 5'-triphosphate (dTTP) in the extension of a deoxyribo 17-mer primer by Sequenase, a modified T7 DNA polymerase, using a 25-mer template containing one 2'-deoxyadenosine residue. No detectable pause in polymerization was found at the dTTP incorporation site. These findings suggest that thymidine 5'-0-(1-boranotriphosphate), possibly one of the two phosphorus diastereoisomers like the (Sp) diastereoisomer of the analogous thiotriphosphates 4(Burgers, P. M. J.; Eckstein, F., J. Biol. Chem. 1979, 254, 6889-6893 and Romaniuk, P. J.; Eckstein, F., ibid 1982, 257, 7684-7688) is a substrate for polymerases.
It was also observed that acid phosphatase from sweet potato (EC 3.1.3.2) and 5'-nucleotidase from Crotalus adamanteus venom (EC 3.1.3.5) completely hydrolyses thymidine 5'-boranophosphate to thymidine. On the other hand, thymidine 5'-boranophosphate is a very poor substrate (or an inhibitor) of alkaline phosphatase from Eschrichia coli (EC 3.1.3.1). The analogous thiophosphate, thymidine 5'-thiophosphate, is a competitive inhibitor of both acid and alkaline phosphatases. The fact that thymidine 5'-boranophosphate is a substrate of acid phosphatase, while thymidine 5'-thiophosphate is a competitive inhibitor of the same enzyme, is remarkable and demonstrates the potential for the complimentary use of boranophosphates and thiophosphates to study the details of the mechanism of enzymic reactions.
In addition to molecular biology studies, modified nucleosides and nucleotides have demonstrated considerable pharmacological activity in the antiviral and antitumor areas (Mitsuya, H. Broder, S., Proc. Natl. Acad. Sci. USA, 1986, 83, 1911-1915; Mitsuya, H. et al, Proc. Natl. Acad. Sci. USA 1985, 82, 7096-7100; Lin, T. S. et al, J. Med. Chem., 1988, 31, 336-340; Beauchamp, L. M. et al, J. Med. Chem. 1988, 31, 144-149; Remy, R. J., Secrist III, J. A., Nucleosides Nucleotides 1985, 4, 411-427; Prusoff, W. H., Ward, D. C., Biochem. Pharmacol., 1976, 25, 1233-1239; Marquez, V. E. et al, J. Med. Chem. 1988, 31, 1687-1694; Lin, T.-S., Prusoff, W. H., J. Med. Chem. 1978, 21, 109-112; Johnson, F. et al, J. Med. Chem. 1984, 27, 954-958; Secrist et al, J. Med. Chem. 1988, 31, 405-410; Farquherz, D., Smith, R., J. Med. Chem. 1985, 28, 1358-1361; Hunston, R. H. et al, J. Med. Chem. 1984, 27, 440-444; Farquher, D. et al, J. Med. Chem. 1983, 26, 1153-1158; McGuigan et al, Nucleic Acids Res. 1989, 17, 6065-6075; McGuigan et al, Nucleic Acids Res. 1989, 17, 10171-10177; Colin, B. et al, Nucleic Acids Res. 1989, 17, 7195-7201 and Lambert et al, J. Med. Chem. 1989, 32, 367-374). Thus, phosphite-boranes with nucleoside substituents may combine the pharmacological properties of Lewis-base-borane compounds and those of modified nucleosides to give superior therapeutic agents.
While it is clear that considerable potential exists for the utility of phosphite-borane derivatives with nucleoside substituents as biomolecular probes and therapeutic agents, it is equally dear that not much effort has been focused on exploiting this potential. The present invention arose from our ongoing research into boron analogs of biomolecules potentially useful as probes and therapeutic agents.
It therefore is an object of the present invention to provide new phosphite-borane derivatives including active antineoplastic, anti-hyperlipidemic, and anti-inflammatory agents.
It is another object of the present invention to provide new processes for synthesizing phosphite-borane derivatives exhibiting antineoplastic, anti-hyperlipidemic, and anti-inflammatory activity.
Other objects and advantages will be more fully apparent from the ensuing disclosure and appended claims.